Factor H-Fc immunotheraphy

ABSTRACT

Fusion proteins comprising human FH domains 18-20 (FH18-20) linked via an optional linker to IgG Fc, wherein the FH has mutation of D to G at position 1119 in domain 19; FHD1119G/Fc), and methods of use thereof, e.g., to treat pathogen infections.

CLAIM OF PRIORITY

This application is a national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/US2016/059072, filed on Oct. 27, 2016,which claims the benefit of U.S. Provisional Patent Application Ser. No.62/246,662, filed on Oct. 27, 2015. The entire contents of each of theforegoing applications are hereby incorporated by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant Nos.AI111728, AI118161, AI054544 and AI032725 awarded by the NationalInstitutes of Health. The Government has certain rights in theinvention.

TECHNICAL FIELD

Described herein are fusion proteins comprising Factor H (FH) domains18-20 (FH18-20) linked via an optional linker to IgG Fc, wherein the FHhas mutation of D to G at position 1119 in domain 19; FHD1119G/Fc), andmethods of use thereof, e.g., to treat pathogen infections.

BACKGROUND

Antimicrobial resistance remains a major threat to public healthworldwide and we are witnessing an era where several medically importantmicrobes are becoming untreatable with antibiotics currently in clinicaluse. Organisms such as Staphylococcus aureus, Enterococcus spp,Pseudomonas aeruginosa, Acinetobacter baumanii, and several additionalmembers of the family Enterobacteriaceae have become resistant to mostconventional antibiotics (1, 2). Similarly, Neisseria gonorrhoeae hasalso demonstrated a remarkable capacity to resist almost everyantibiotic that it has encountered (3). The recent isolation of N.gonorrhoeae strains resistant to ceftriaxone, the last remaining optionfor empirical monotherapy, in several parts of the world represents amajor public health problem (4-7). In addition to complicationsincluding pelvic inflammatory disease and its sequelae such asinfertility, ectopic pregnancy and chronic pelvic pain, gonorrhea canincrease the transmission and acquisition of HIV-1 infection (8, 9).Thus, spread of multidrug resistant gonorrhea represents a seriouspublic health threat and there is an urgent need to develop novelantimicrobials and (ideally) vaccines and immunotherapeutics againstthis pathogen.

SUMMARY

Neisseria gonorrhoeae (Ng), the causative agent of the sexuallytransmitted infection gonorrhea, has developed resistance to almostevery conventional antibiotic. There is an urgent need to develop noveltherapies against gonorrhea. Many pathogens, including Ng, bind thecomplement inhibitor factor H (FH) to evade complement-dependentkilling.

Chimeric proteins comprising human FH domains 18, 19 and 20 fused tomurine IgG2a Fc (FH18-20/Fc) bound to gonococci, activated the classicalpathway of complement, and resulted in complement-dependent bactericidalactivity (27). Such a molecule could serve as a novel adjunctiveimmunotherapeutic against multidrug-resistant bacterial species,including N. gonorrhoeae. However, the C-terminus of FH is also criticalfor regulating complement activation on host cells (28,29). Therefore, atherapeutic that uses the C-terminus of FH to anchorcomplement-activating Fc to the bacterial surface needs to be modifiedto eliminate binding to host cells.

Sialylation of gonococcal lipooligosaccharide (LOS), as occurs in vivo,augments binding of human FH through its domains 18-20 (FH18-20). Weexplored the utility of fusing FH18-20 with IgG Fc (FH18-20/Fc) tocreate a novel anti-infective immunotherapeutic. FH18-20 also binds toselect host glycosaminoglycans to limit unwanted complement activationon host cells. To identify mutation(s) in FH18-20 that eliminatedcomplement activation on host cells, yet maintained binding to Ng, wecreated four mutations in domains 19 or 20 described in atypicalhemolytic uremic syndrome that prevented binding of mutated fH to humanerythrocytes. One of the mutant proteins (D to G at position 1119 indomain 19; FHD1119G/Fc) facilitated complement-dependent killing ofgonococci similar to unmodified FH18-20/Fc, but unlike FH18-20/Fc, didnot lyse human erythrocytes. FHD1119G/Fc bound to all (100%) of 15sialylated clinical Ng isolates tested (including three contemporaryceftriaxone-resistant strains), mediated complement-dependent killing of10/15 (67%) strains and enhanced C3 deposition (>10-fold above baselinelevels) on each of the five isolates not directly killed by complement.FHD1119G/Fc facilitated opsonophagocytic killing of a serum-resistantstrain by human polymorphonuclear neutrophils. FHD1119G/Fc administeredintravaginally significantly reduced the duration and burden ofgonococcal infection in the mouse vaginal colonization model.FHD1119G/Fc represents a novel immunotherapeutic againstmultidrug-resistant Ng.

Thus, in a first aspect the invention provides polypeptides comprisingFactor H (FH) domains 18-20 (FH18-20) linked via an optional linker toIgG Fc, wherein the FH18-20 has mutation at position 1119 in domain 19,and wherein the linker comprises at least two additional amino acidsbetween the FH domain and the Fc region.

In some embodiments, the Fc region is derived from a humanimmunoglobulin or a murine immunoglobulin.

In some embodiments, the linker comprises at least one glycine or onealanine. In some embodiments, the linker comprises GAAGG (SEQ ID NO:1)or AAAGG (SEQ ID NO:2).

In some embodiments, the polypeptide further comprises a peptide tag,e.g., hemagglutinin (HA), FLAG, HIS, c-Myc, VSV-G, V5, or HSV.

In some embodiments, the FH18-20 has a mutation of D to G at position1119 in domain 19.

Also provided herein are pharmaceutical compositions comprising fusionproteins described herein, and a pharmaceutically acceptable carrier,e.g., for use in the treatment of a disorder associated with a FactorH-binding pathogen.

Further, provided herein is the use of a fusion protein described hereinin the manufacture of a medicament for the treatment of a disorderassociated with a Factor H-binding pathogen. In addition, describedherein are methods for treating disorders associated with a FactorH-binding pathogen in a subject, the method comprising administering atherapeutically effective amount of a fusion protein described herein.

In some embodiments, the disorder is infection with N. gonorrhoeae, andthe methods include administering a therapeutically effective amount ofa fusion protein as described herein.

In some embodiments, the disorder is a pathogen-associated infection.

In some embodiments, the pathogen is selected from the group consistingof bacteria, fungi, viruses, spirochetes, and parasites.

In some embodiments, the bacterium is selected from the group consistingof P. aeruginosa, S. pneumoniae, Y. pestis, E. coli, S. typhimurium, N.meningitidis, N. gonorrhoeae, H. influenza and S. aureus.

In some embodiments, the fungus is selected from the group consisting ofAspergillus fumigatus, Candida albicans, and other zymosan-containingfungi.

In some embodiments, the spirochete is Borrelia burgdorferi or Treponemapallidum.

In some embodiments, the parasite is Plasmodium berghei or Plasmodiumfalciparum.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-C. Binding to, and bactericidal activity against N. gonorrhoeaeof wild-type FH18-20/murine IgG2a Fc and its derivatives containingpoint mutations in the FH region. A. Binding of WT FH18-20/murine IgG2aFc and mutant proteins to N. gonorrhoeae strain F62 grown in mediacontaining CMP-Neu5Ac (2 μg/ml) to sialylate its LOS. The D1119Gmutation is located in FH domain 19; R1182S, W1183R and R1215G mutationsare in FH domain 20. Each FH/Fc protein (10 μg/ml) was incubated withsialylated strain F62 and bound FH/Fc was detected by flow cytometry.Numbers alongside each histogram represents the median fluorescence ofthe entire bacterial population. The control reaction mixture lacksFH/Fc. B. Bactericidal activity of FH18-20/murine IgG2a Fc and mutantproteins directed against sialylated N. gonorrhoeae. Sialylated strainF62 (left graph) or sialylated strain 252 (right graph) were incubatedwith varying concentrations of each FH/Fc molecule (X-axis), followed bythe addition of human complement (IgG/IgM depleted human serum [seeMethods]). C. Survival of bacteria at 30 min relative to bacterialcounts at the beginning of the assay (to min) is shown on the Y-axis(mean±SD of at least 2 independently performed experiments).

FIG. 2. Complement-mediated lysis of anti-CD59 treated RBCs by FH/Fcfusion proteins. Increasing concentrations (indicated on the X-axis) ofwild type FH18-20/murine IgG2a Fc or FHD1119G/Fc (either murine IgG2a Fcor human IgG1 Fc) were added to anti-CD59-treated human RBCs inGVB/Mg⁺⁺EGTA followed by the addition of homologous NHS to aconcentration of 40%. The reaction mixture was then incubated at 37° C.for 20 min. Cold GVB/EDTA was added, samples were centrifuged andOD_(410 nm) was measured to determine degree of hemolysis (indicated onthe Y-axis). Each experiment was performed in duplicate and resultsindicated and each point represents the mean (SD).

FIG. 3. Binding of FHD1119G/human Fc to strains of sialylated N.gonorrhoeae. Binding of FHD1119G/human IgG1 Fc (10 ug/ml) was measuredby flow cytometry. Y-axis represents median fluorescence (mean [SD] of 2independently performed experiments). ‘Control’ represents a reactionmixture lacking FH/Fc. Strains that are resistant to ceftriaxone(MIC>0.25 μg/ml) are indicated by solid black bars, strains that showelevated MICs to ceftriaxone (MICs 0.125 ug/ml and 0.25 ug/ml) byhatched bars and ceftriaxone-sensitive isolates (MICs<0.125 ug/ml) bygrey shaded bars.

FIG. 4. Complement-dependent bactericidal activity of FHD1119G/Fcagainst N. gonorrhoeae. Sialylated N. gonorrhoeae strains (same strainsas in FIG. 4) were incubated with FHD1119G/human IgG1 Fc (33.3 μg/ml;grey bars) or buffer alone (controls; black bars), followed by theaddition of 20% (v/v) human complement for 30 min at 37° C. Percentsurvival of bacterial counts at 30 min relative to counts at thebeginning of the assay (to min) is shown on the Y-axis (mean±SEM of atleast 2 separately performed experiments). Strains that resisted killingby FHD1119G/Fc plus complement (>50% survival of CFU at 30 min) areindicated (“#”).

FIG. 5. C3 fragment deposition on 5 sialylated strains that resistedkilling by complement: FA1090, CTXr (Spain); NJ-11; NJ-19 and NJ-26(indicated by “#” in FIG. 4). Sialylated strains were incubated withFHD1119G/human IgG1 Fc (33.3 μg/ml) and 20% (v/v) human complement.Sialylated F62 served as a positive control. C3 fragments (C3b/iC3b)deposited on bacteria in the presence of FHD1119G/Fc were detected byflow cytometry (histograms shown with a solid black line). C3 fragmentdeposition on bacteria incubated with complement alone shown by the greyshaded histogram and antibody conjugate controls (bacteria plus anti-C3cFITC) by the dashed histogram. The number next to each histogramrepresents median fluorescence. One representative experiment of tworeproducible repeat experiments is shown.

FIG. 6. Opsonophagocytic killing of the sialylated Opa-negative mutantof N. gonorrhoeae FA1090 by FHD1119G/human Fc and complement.Opa-negative FA1090 grown in media containing CMP-Neu5Ac to sialylatelipooligosaccharide (LOS)(10⁷ CFU) was incubated with FHD1119G/human Fc(FHD1119G/HuFc; 16.7 μg/ml) and 20% (v/v) human complement, followed bythe addition of 10⁶ freshly isolated human PMNs for 60 min at 37° C.(MOI 10:1). Bacterial survival at 60 min relative to to is shown on theY-axis (mean [SD] of 4 independently performed experiments). Controlsincluded reactions where complement was heat-inactivated (indicated by“-” in the “Active complement” row), or where FHD1119G/Fc was omitted.*, P<0.05; **, P<0.01 (ANOVA).

FIGS. 7A-C. FHD1119G/mouse IgG2a Fc reduces the duration and burden ofgonococcal infection in the murine vaginal model of gonococcalcolonization. Two groups of Premarin-treated wild-type BALB/c mice wereinfected with 1.5×10⁶ CFU of N. gonorrhoeae strain F62 and given either12 μg FHD1119G/mouse IgG2a Fc (n=14) or a corresponding volume of PBS(n=12) as a vehicle control. Each mouse also received 10 μg ofCMP-Neu5Ac daily as described in the Materials and Methods. Vaginalswabs were obtained daily to quantify Ng CFUs. A. Kaplan Meier analysisof time to clearance. B. Colonization of bacteria (log₁₀ CFU) measureddaily. C. Bacterial burdens consolidated over time (Area Under the Curve[log₁₀ CFU] analysis) for the two groups.

FIG. 8. FHD1119G/human IgG1 Fc and FHD1119G/mouse IgG2a Fc havecomparable bactericidal activities. Survival of sialylated N.gonorrhoeae F62, H041 and NJ-60 in increasing concentrations of FHD1119Gfused to either human IgG1 Fc (grey bars) or mouse IgG2a Fc (black bars)was determined following the addition of 17% (v/v) pooled normal humanserum (NETS; not depleted of natural Ab). Percent survival of bacteriaat 30 min relative to to is shown on the Y-axis (mean [SD] of 2separately performed experiments). Reaction mixtures containing bacteriaand pooled NETS are indicated by hatched bars.

FIG. 9. Representative histogram tracings of binding of FHD1119G/humanIgG1 Fc to 15 clinical isolates of N. gonorrhoeae sialylated in vitro.Each of the 15 isolates shown above was incubated with FHD1119G/humanIgG1 Fc (10 μg/ml) and bound FH/Fc was detected by flow cytometry usinganti-human IgG FITC (grey shaded histograms). Controls, shown by thehistograms with the broken lines, represent reaction mixtures lackingFH/Fc. The numbers alongside the histograms represents the medianfluorescence of the entire bacterial population.

FIGS. 10A-C. Efficacy of FHD1119G/Fc against Ng FA1090. BALB/c mice(n=10/group) were infected with ˜10⁶ CFU of Ng and treated dailyintravaginally with PBS (vehicle control) or FHD1119G/human IgG1 Fc (10μg/day). Vaginal swabs were obtained daily to enumerate viable CFUs. A.Kaplan Meier curve shows time to clearance (Mantel-Cox analysis). B.Log₁₀ CFU versus time is shown. C. Bacterial burdens consolidated overtime (Area Under Curve (log₁₀ CFU) analysis are shown (comparisons madeby Mann-Whitney U test).

FIGS. 11A-C. Efficacy of FHD1119G/Fc against ceftriaxone-resistant(CRO-R) strain H041. BALB/c mice (n=10/group) were infected with ˜10⁶CFU of Ng and treated daily intravaginally with PBS (vehicle control) orFHD1119G/human IgG1 Fc (10 μg/day). Vaginal swabs were obtained daily toenumerate viable CFUs. A. Kaplan Meier curve shows time to clearance(Mantel-Cox analysis). B. Log₁₀ CFU versus time is shown. C. Bacterialburdens consolidated over time (Area Under Curve (log₁₀ CFU) analysisare shown (comparisons made by Mann-Whitney U test).

DETAILED DESCRIPTION

Resistance of pathogens to many of the currently available antimicrobialagents poses a major threat to human health worldwide. The Centers forDisease Control and Prevention (CDC) has proclaimed that N. gonorrhoeaeis one of three organisms (together with Clostridium difficile andcarbapenem-resistant Enterobacteriaceae) where resistance toantimicrobials represents an “urgent threat” to human health (5). The“Global action plan to control the spread and impact of antimicrobialresistance in Neisseria gonorrhoeae” recently published by the WHOemphasizes the need for novel approaches to prevent and treat gonorrhea(54). Newer modalities of treatment whose mechanism(s) of action differfrom those of conventional agents provide hope that drug-resistance maybe deterred when traditional mechanisms of selection are circumvented(3).

The complement system forms a key arm of innate immune defenses againstinvading pathogens (10). In order to successfully establish infectionsin their hosts, microbes have developed mechanisms to subvert killing bycomplement (11). By binding of complement inhibitors, such as factor H(FH), C4b-binding protein (C4BP) and vitronectin, several pathogens,including N. gonorrhoeae, dampen complement activation on their surfaces(11-13). FH inhibits the alternative pathway of complement by serving asa cofactor for the factor I-mediated cleavage of C3b to thehemolytically inactive iC3b fragment (14). FH also possesses decayaccelerating activity, whereby it irreversibly dissociates the Bbfragment from the alternative pathway C3 convertase, C3b,Bb (15-17). FHcomprises 20 domains, also known as short consensus repeat domains(SCRs) or complement control protein domains (CCPs) that are arranged inthe form of a single chain (18). The first four N-terminal domains arenecessary and sufficient for complement inhibition (19). Most microbes,including N. gonorrhoeae, that bind FH do so through regions spanned bydomains 6 and 7 and/or domains 18 through 20 (11).

Sialylation of gonococcal LOS is an important component of gonococcalpathogenesis, which occurs in humans (20, 21) and also duringexperimental infection of mice (55). In vivo, gonococci scavenge5′-cytidinemonophospho-N-acetylneuraminic acid (CMP-Neu5Ac) from thehost to sialylate their lipooligosaccharide (LOS) (20, 21). The two LOSstructures that can be sialylated are the nearly ubiquitously expressedlacto-N-neotetraose (LNT, Neu5Acα2-3Galβ1-4G1cNAcβ1-3Galβ1-4G1cβ1-4HepI)structure and the less frequently encountered P^(k)-like structure(Neu5Acα2-6Galα1-4Galβ1-4G1cβ1-4HepI) (22). Sialylation of the LNT LOSstructure enhances binding of the C-terminal domains 18-20 of human FHto gonococci (23, 24). This increase in FH binding is dependent onexpression of gonococcal porin (PorB); replacing gonococcal PorB withmeningococcal PorB abrogates Neu5Ac-mediated enhancement of FH binding(25). Several strains of N. gonorrhoeae also bind FH independently ofLOS sialylation (26). Gonococcal mutants that are incapable of LOSsialylation following deletion of the LOS sialyltransferase (1st) geneare less virulent in the mouse model of vaginal colonization (55).Sialylation of LOS facilitates evasion of gonococcal killing by thealternative and classical pathways of complement and may also augmentbacterial resistance to killing by cationic peptides (56).

LOS sialylation enhances FH binding through C-terminal domains of FH(24, 27). In addition, a chimeric molecule comprising FH domains 18-20fused to mouse IgG2a Fc mediates complement-dependent killing ofsialylated gonococci (27). Killing of gonococci by FH/Fc is classicalpathway dependent and occurs at Fc concentrations well below thatrequired to block FH binding to bacteria (27). However, because theC-terminal domains of FH plays a key role in “self-nonself”discrimination (28, 29), the use of a FH18-20/Fc molecule with anunmodified FH has the capacity to bind to human cells and activatecomplement; this is revealed in our experiments of complement-dependentlysis of anti-CD59-treated RBCs by unmodified FH18-20/Fc (FIG. 2). FHdomains 19 and 20 interact with C3 fragments and glycosaminoglycansrespectively, to limit complement activation on host cells (28, 29),Therefore it was necessary to introduce a mutation in the FH fragment toabrogate toxicity. To achieve this, we capitalized on prior work thatcharacterized select mutations in FH domains 19 and 20 that have beendescribed in individuals with aHUS (37). We focused on four FH mutationsthat did not interfere with full-length FH's inhibition of lysis ofhuman erythrocytes and selected a mutant, FHD1119G/Fc that showedactivity that was comparable to FH18-20/Fc activity against gonococcibut did not exhibit complement-dependent lysis of human RBCs.

It is worth noting that although FHD1119G/Fc did not cause directcomplement-mediated killing of five of 15 tested isolates (FIG. 4), itenhanced C3 deposition on all of these five isolates (FIG. 5) andresulted in opsonophagocytic killing of an Opa-negative mutant derivedfrom one of these strains (FIG. 6). The reason(s) for the resistance ofthese five strains to direct complement-dependent killing despite theobserved enhanced C3 deposition is not clear. Possible (and not mutuallyexclusive) explanations include insufficient C5 convertase formationand/or prevention of effective C5b-9 formation, for example by bindingvitronectin (57-60). The relative roles of membrane attackcomplex-mediated bacterial killing versus opsonophagocytosis inclearance of gonococci in vivo remains to be elucidated.

FHD1119G/Fc showed activity against gonococci in the mouse vaginalcolonization model and represents a promising initial step in the searchfor novel therapeutics against gonorrhea that is rapidly becomingmultidrug-resistant. We acknowledge that further studies to evaluate thesafety of FH/Fc as well as its efficacy against other strains ofgonorrhea are needed.

Notably, Meri et al (61) showed that the D1119G mutation in FH domain 19did not affect binding to several microbes, including Pseudomonasaeruginosa, Haemophilus influenzae, Bordetella pertussis, Streptococcuspneumoniae and Candida albicans, suggesting that FHD1119G/Fc may alsoenhance complement activation and possess therapeutic activity againstthese pathogens. In particular, P. aeruginosa and C. albicans have beencited by the CDC as microbes where drug-resistance represents a“serious” threat level (5). Activity of FHD1119G/Fc as an adjunctivetreatment in these infections merits study.

In this study we have focused on LOS sialylation, a key virulencemechanism of gonococci, to design a novel FH/Fc fusion protein thatpossesses bactericidal activity (either direct killing by complement orthrough opsonophagocytosis) against a wide array of gonococcal isolatesin vitro. In order to develop resistance to this agent, gonococci wouldhave to lose the ability to sialylate LOS and bind to FH; decrease inresistance to complement and cationic peptides would result indiminished fitness and pose a barrier to the development ofdrug-resistance, which may not be simply overcome by the traditionalmicrobial mechanism of “escape mutation” (3). Accordingly, gonococcithat lack the ability to sialylate its LOS (1st deletion mutant) areout-competed by the parent strain in the mouse vaginal colonizationmodel (55, 62).

Here we describe derivation of a novel fully human FH18-20/Fc fusionimmunotherapeutic molecule (FHD1119G/Fc) that shows activity both invivo and in vitro against diverse N. gonorrhoeae isolates, and which mayalso be active against Pseudomonas aeruginosa, Haemophilus influenzae,Bordetella pertussis, Streptococcus pneumoniae and Candida albicans,among other pathogens.

Factor H

Factor H is a complement-inhibitory molecule whose main roles are tolimit the amount of C3b deposited on a surface and also facilitate theconversion of active C3b to the hemolytically inactive molecule, iC3b,and thus limit complement activation that prevents activation of thelytic effector system. The binding of Factor H to the surface of somebacteria confer them a “protective” effect against the C-dependentlysis. Factor H pathogen recognition molecules can be derived from,e.g., Homo sapiens (GenelD: 3075; UniGene Hs.363396; NCBI Accession#NP_000177.2 or P08603). In some embodiments, the Factor H PRM includesCCP/Sushi domains 18-20 described in Table A; the numbers refer to theamino acids of NCBI Accession #NP_000177.2.

TABLE A CCP/Sushi domains 18-20 of human complement factor H CCP/Sushi18 1046 1104 CCP/Sushi 19 1107 1165 CCP/Sushi 20 1170 1230

The Factor H domains used in the fusion proteins described hereininclude at least one mutation, a mutation of D at position 1119 indomain 19, referred to herein as FH(18-19)D1119X. In some embodiments,the mutation is a D to G mutation, although other amino acids can alsobe substituted for the D (referred to herein as FH(18-19)D1119G), e.g.,Alanine (A), isoleucine (I), Leucine (L), Proline (P), or Serine (S).

In some embodiments, the FH(18-19)D1119X comprises SEQ ID NO:3; theposition 1119 is shown here as a bold, double underlined “X”:

(SEQ ID NO: 3) CVNPPTVQNAYIVSRQMSKYPSGERVRYQCRSPYEMFGDEEVIVICLNGNWTEPPQCKDSTGKCGPPPPIDNG X ITSFPLSVYAPASSVEYQCQNLYQLEGNKRITCRNGQWSEPPKCLHPCVISREIMENYNIALRWTAKQKLYSRTGESVEFVCKRGYRLSSRSHTLRTTCWDGKLEYPTCAKR

Fc Modules

The mouse and human immunoglobulin (IgG) heavy chain has four Ig-likedomains termed V_(H) (Variable heavy) and C_(H1) (Constant heavy 1) toC_(H3) (Constant heavy 3). A “hinge” region separates the C_(H1) andC_(H2) domains. The hinge region contains a variable number of cysteineresidues (three in the mouse IgG_(2a)) that can form covalent interchainbonds between two identical immunoglobulin heavy chains. The portion ofan immunoglobulin comprising the hinge region plus the domains C_(H2)and C_(H3) is called fragment crystallizable (Fc). There are severaldifferent human and other mammalian (e.g., murine) IgG molecules. Forexample, the human equivalent of mouse IgG2a is the IgG1. Severalimmunotherapeutic agents for human therapy include the human IgG1 Fcportion. A portion of the Fc molecules is used to prepare the Fc portionof the chimeric fusion protein molecules described herein.

The fusion proteins can contain sequences from the same species or fromdifferent species. For example, an interspecies hybrid fusion proteincan contain a murine Fc region and a human sequence from an FH protein.The fusion proteins described herein also preferably fully human (i.e.,a human FH 18-20 domain and a human Fc region). In general, both the FHmodule and the Fc region of a fusion protein for use in a specificanimal species are derived from that animal species. Thus, a humanFH(18-20):human Fc fusion protein is generally used in humans.

General methods of preparing fusion proteins are known in the art(Ashkenazi, A. and S. M. Chamow (1997), “Fusion proteins as researchtools and therapeutic agents,” Curr. Opin. Immunol. 9(2): 195-200,Chamow, S. M. and A. Ashkenazi (1996). “Fusion proteins: principles andapplications,” Trends Biotechnol. 14(2):52-60). In general, to generatea fusion protein, the sequences encoding the hinge region of an Ig areretained and a region coding for a short (e.g., about 5 amino acid)linker is added between the pathogen recognition module coding regionand the region coding for the Fc (n-terminal to the hinge). The maineffector region of the Fc (i.e., the region that binds complement andprotein A, and the single glycosylation site that is required tostabilize an Fc dimer—the effector functions are C-terminal to the hingeregion) should be included.

In one example, a fusion protein can be made by cloning into anexpression vector such as pcDNA3 (Invitrogen) a nucleic acid sequenceencoding a TLR ECD in-frame with a sequence encoding an Fc portion of anIg (e.g., the Fc portion of an IgG such as an IgG2a).

In one embodiment, the Fc portion and a linker (in bold print below) hasthe murine sequence:

(SEQ ID NO: 4) AAAGGEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK.

In other embodiments, the Fc portion can be derived from the human Iggamma-1 chain C region (Swiss-Prot Accession No. P01857), in which thehinge starts from residue 99:

(SEQ ID NO: 5) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR[D/E]E[L/M]TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In some embodiments, the Human IgG1 sequence used is:

(SEQ ID NO: 6) EPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Linkers

In some embodiments, the fusion protein construct includes a linker,e.g., in the form of additional residues, e.g., alanine and/or glycineresidues, between the FH(18-20) and the Fc/Ig hinge region. The totalnumber of linker residues (in addition to glycine residues that arenaturally occurring in the Ig from which the hinge region is derived)can be, e.g., at least 2, 3, 4, 5, 6, or 7. To minimize the possibilityof immunological rejection of the molecule and retain expression andproper folding other residues can be used. These include naturallyoccurring Ig hinge regions or part of non-structured regions of humanextracellular proteins. As a general rule, when designing a Fusionprotein hinge region, peptide sequences including small, slightlyhydrophilic amino acids such as glycine, alanine, serine, threonine,methionine are preferred over charged, ring or aromatic residues. Thus,the total number of resides, e.g., alanine and/or glycine residues inthe linker region can be, e.g., at least 2, 3, 4, 5, 6, 7, 8, or 9.Examples of linkers include GAAGG (SEQ ID NO:1) and AAAGG (SEQ ID NO:2).These examples are not to be construed as limiting and in general, alinker that results in a fusion protein that can bind to its cognateligand is encompassed by the invention. In some embodiments, the nucleicacid sequence that encodes the linker includes a restriction enzymerecognition site, e.g., Not I, to facilitate generation of fusionprotein constructs.

Fusion Proteins

The methods and compositions described herein can be used to make fusionproteins that are highly purified. Such highly purified proteins can beused, e.g., in a method of treating a subject who has an infection.

Constructs encoding fusion proteins can be transfected into a cell usingmethods known in the art. The cells can be cultured under conditionssuitable for expression of the cloned fusion protein. Suitable cellsinclude HEK293 (human), COS7 (monkey), and CHO (hamster) cells, althoughfor production purposes, any eukaryotic cell type that can be engineeredto produce a correctly folded and glycosylated fusion protein ofinterest can be used, including insect expression systems. In general,cells that produce antibodies (e.g., B cells) are not used.

The fusion protein vector or construct (a vector that encodes a fusionprotein) can be further engineered such that a secretory signal is partof the fusion protein. Methods are known in the art for engineering anucleic acid sequence to encode a secretory signal such that a fusionprotein is secreted or embedded in the membrane. An inducible promotercan also be positioned to control the expression of the fusion proteinso that expression of the fusion protein can be induced. Examples ofsuch inducible promoters include a metallothionein promoter, atetracycline sensitive promoter (tet- on tet-off), or a copper-induciblepromoter. In addition, a fusion protein vector can have a retroviralbackbone and/or include a gene that confers antibiotic resistance to acell. Thus, transfected or transduced cells can be selected using theantibiotic to which the gene encodes a resistance protein to select fora stable transgene.

Fusion proteins can be detectably labeled for various uses such as thosedescribed herein. Labeled fusion proteins (such as fusion proteins) canbe used, for example, as commercially produced reagents for use inFactor H assays and in methods for identifying compounds that bind toFactor H.

Examples of detectable labels include various enzymes, prostheticgroups, fluorescent materials, luminescent materials, bioluminescentmaterials, and radioactive materials. Examples of suitable enzymesinclude horseradish peroxidase, alkaline phosphatase, β-galactosidase,or acetylcholinesterase; examples of suitable prosthetic group complexesinclude biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate (FITC),rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride orphycoerythrin (PE); examples of bioluminescent materials includeluciferase (which oxidates luciferin or luminol, producing light as abyproduct), luciferin, luminol and aequorin, and examples of suitableradioactive material include ¹²⁵I, ¹³¹I, ³⁵S, ³²P, ³H. Methods oflinking such molecules to a polypeptide are known in the art.

In some embodiments, a fusion protein is labeled by including anadditional moiety such as a FLAG epitope in the hybrid protein (e.g., byengineering a vector that encodes desired fusion protein-FLAG hybridprotein), a fluorescent protein like the green fluorescent protein andits spectrum variants, or by coupling (e.g., covalently linking) adetectable moiety such as a fluorescent molecule to the fusion protein.

Assays for Fusion Protein Activity

The fusion proteins described herein have one or more of the followingactivities: (1) inhibit bacterial proliferation, (2) triggercomplement-mediated cytotoxicity, and/or (3) function as an artificialopsonin. For example, some fusion proteins might bind to and killbacteria, but not activate complement deposition; other fusion proteinsmight enhance phagocytosis, but not activate complement, and vice-versa.Methods are described herein that can be used, e.g., to evaluate afusion protein to measure the efficiency with which the fusion proteinneutralizes the pathogens to which it binds, e.g., by these three modesof action.

In some embodiments, the methods described herein include applying afusion protein to a test sample including a cell or living tissue ororgan, and evaluating one or more activities of the fusion protein,e.g., the ability of the fusion protein to bind and/or activatecomplement, and/or to bind a pathogen and trigger opsonophagocytosis.

In some embodiments, the test sample is, or is derived from (e.g., asample originally taken from) an in vivo model of a disorder asdescribed herein. For example, an animal model, e.g., a rodent such as arat, that is infected with a pathogen can be used, and the ability ofthe fusion protein to improve one or more symptoms of the disorder,e.g., clinically relevant symptoms, is evaluated.

Methods for evaluating each of these effects are known in the art; someare described herein.

A test compound that has been screened by a method described herein anddetermined to be active, e.g., to bind and activate complement, and/orto bind a pathogen and trigger opsonophagocytosis, can be considered acandidate compound. A candidate compound that has been screened, e.g.,in an in vivo model of a disorder, e.g., an animal infected with apathogen, e.g., a microbe, and determined to have a desirable effect onthe disorder, e.g., on one or more symptoms of the disorder, can beconsidered a candidate therapeutic agent. Candidate therapeutic agents,once screened in a clinical setting and found to be effective, aretherapeutic agents. Candidate compounds, candidate therapeutic agents,and therapeutic agents can be optionally optimized and/or derivatized,and formulated with physiologically acceptable excipients to formpharmaceutical compositions.

Opsonophagocytosis Assays

Phagocytosis is an important mechanism of bacteria killing and clearancefrom the site of infection. Fusion proteins might play an important roleas opsonins in addition to their direct role in activating complement.Fusion proteins are chimeric proteins that contain the immunoglobulin Fcdomain and preliminary studies demonstrated that they can bind Fcreceptors on macrophages. When fusion proteins coat bacteria, they willlikely provide anchorage sites to the Fc receptors on the surface ofphagocytes and promote the Fc receptor-mediated phagocytosis of thebacterial particles. These internalized fusion protein-coated particleswould be decomposed intracellularly and the components would be directedto the antigen “presentation” machinery. In addition, shed fusionprotein ligands might directly enter the presentation pathways via Fcreceptor internalization, thus enhancing their presentation. Eitheroutcome would be of pivotal importance for the healing process and theestablishment of an immune memory.

Fusion protein-mediated opsonization might trigger bacterial killing viaMAC (membrane attack complex) deposition on their cell walls, whilepromoting phagocytosis and cell mediated killing by professionalphagocytes. The efficiency of fusion proteins as artificial opsonins canbe measured by evaluating enhanced opsonophagocytosis and antigeninternalization in vitro. For example, two mechanisms of bacterial entryinto cells in vitro can be evaluated: 1) uptake by “non-professional”phagocytes such as the HEK293 human embryonic kidney cell line and 2)uptake by the macrophage-like cell lines THP-1 and RAW and by humanmacrophages. With “non-professional” phagocytes such as HEK293 cells,bacterial binding to cells that have been transfected with differentfluorescence-tagged Fc receptors can be visually followed.

We have established stably transduced cell lines expressing CD36 taggedwith yellow fluorescence protein (YFP) or CD16 tagged with cyanfluorescence protein (CFP). Both receptors can be visualized in livingcells by confocal microscopy, e.g., using an inverted confocalmicroscope equipped with four laser beams (including a pulse laser forFLIM analysis) and a warmed stage. Confocal microscopy can be used tofollow the formation of Fc receptor clusters around fusionprotein-treated bacteria. The experiments can be conducted underprotein-free conditions to minimize interference from serum components.Bacteria are expected to bind specifically to the Fc receptors only whenthey are coated with the Fc-containing fusion proteins. With fusionprotein bridging via their Fc portion, a fluorescent “cup” will form atthe interface bacteria/cell membrane. HEK293 cells, which do notnormally internalize bacteria, also might become internalizationcompetent.

To establish whether fusion proteins can enhance phagocytosis inprofessional phagocytes, similar experiments can be performed, e.g.,with macrophage-like cell lines such as THP-1 and RAW, and with humanmacrophages purified from the blood of healthy donors. Cellularinternalization of bacteria that have been coated with fusion proteincan be measured, with uncoated bacteria serving as controls.Commercially available Fc receptor-blocking antibodies can be used todetermine the contribution of fusion protein opsonization. It isexpected that under protein-free conditions, non-professional phagocyteswill efficiently internalize bacteria only if they are coated withfusion protein, whereas professional phagocytes will internalize bothcoated and uncoated bacteria but fusion protein coating will accelerateor enhance bacterial uptake. Bacterial internalization can be measured,e.g., quantitatively by flow cytometry of cells that have been withincubated with fluorescence-tagged bacteria.

Cell mediated killing can be measured by harvesting the cells used forthe phagocytosis assay (or by lysing them directly on plastic afterwashing or killing the non adherent bacteria with antibiotics) anddetermining the number of colony forming units of bacteria from thelysates.

Animal Models

Also included herein are methods of screening compounds by administeringa fusion protein to an animal model of a pathogen-associated disorder.Suitable animal models are known in the art, e.g., mammals, such asmice, rats, or monkeys, infected with a microbe such as Neisseriagonorrhoeae, Pseudomonas aeruginosa, Haemophilus influenzae, Bordetellapertussis, Streptococcus pneumonia or Candida albicans.

The methods include administering at least one dose of a fusion proteinto the animal model, and monitoring the animal for an effect of thecompound on the disorder in the animal, e.g., an effect on a clinicallyrelevant parameter, e.g., a parameter that is related to a clinicalsymptom of the disease as described herein. Methods for selecting,evaluating and scoring such parameters are known in the art.

The animal can be monitored for a change in the disorder, e.g., for animprovement in a parameter of the disorder, e.g., a parameter related toclinical outcome. In some embodiments, the parameter is fever (a trendtowards or a return to normal, e.g., a decrease, would be animprovement); blood pressure (a return to normal, e.g., an increase,would be an improvement); heart rate (a trend towards or a return tonormal, e.g., a decrease, would be an improvement); and respiration rate(a trend towards or a return to normal, e.g., a decrease, would be animprovement); levels of white blood cells (a trend towards or a returnto normal would be an improvement); the level of oxygen (a trend towardsor a return to normal, e.g., an increase, would be an improvement); thenumber of platelets (a trend towards or a return to normal, e.g., anincrease, would be an improvement); lactic acid levels (a trend towardsor a return to normal, e.g., a decrease, would be an improvement); andlevels of metabolic waste products (a trend towards or a return tonormal, e.g., a decrease, would be an improvement).

Pharmaceutical Compositions

A fusion protein can be incorporated into a pharmaceutical composition.Such compositions typically include the fusion protein and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” includes solvents, dispersionmedia, coatings, antibacterial and antifungal agents, isotonic andabsorption delaying agents, and the like, compatible with pharmaceuticaladministration. Supplementary active compounds can also be incorporatedinto the compositions.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude oral or parenteral, e.g., intravenous, intradermal,subcutaneous, inhalation, transdermal (topical), transmucosal, andrectal administration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfate; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, e.g., tromethamine;and agents for the adjustment of tonicity such as sodium chloride ordextrose. pH can be adjusted with acids or bases, such as hydrochloricacid or sodium hydroxide. The parenteral preparation can be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be desirable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate, and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. Such compositionscan also be compounded to minimize exposure to gastric enzymes or tofacilitate uptake by the intestinal tract.

For administration by inhalation, the compounds can be delivered in theform of an aerosol spray, e.g., from a pressurized container ordispenser that contains a suitable propellant, e.g., a gas such ascarbon dioxide, or a nebulizer. Metered dose inhalers are known in theart and can be used. The administration by inhalation can also be usedto treat more than one individual at a time, e.g., to treat an area or anumber of people exposed to a pathogen.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated can be used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents andliposomes. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the active compounds are formulated into ointments, salves, gels, orcreams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery. Such preparationsare particularly useful for treating conditions associated with pathogeninvasion of the lower intestinal tract.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

Oral or parenteral compositions can be provided in dosage unit form forease of administration and uniformity of dosage. Dosage unit form asused herein refers to physically discrete units suited as unitarydosages for the subject to be treated; each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier.

Toxicity and therapeutic efficacy of pharmaceutical compounds containinga fusion protein can be determined by standard pharmaceutical proceduresin cell cultures or experimental animals, e.g., for determining the LD50(the dose lethal to 50% of the population) and the ED50 (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD50/ED50. Compounds that exhibit hightherapeutic indices are preferred. While compounds that exhibit toxicside effects may be used, care should be taken to design a deliverysystem that targets such compounds to the site of affected tissue in tominimize potential damage to non-target cells (e.g., cells that are notundergoing an undesirable inflammatory reaction) and, thereby, reduceside effects. In general, the fusion proteins described herein should bewell-tolerated by an animal (e.g., mouse, non-human primate, or human).

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies generally within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models (e.g., of infection or inflammatory disease)to achieve a circulating plasma concentration range that includes theIC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography or ELISA.

As defined herein, a therapeutically effective amount of a fusionprotein (i.e., an effective dosage) is an amount sufficient to exert atherapeutically beneficial effect. One in the art will appreciate thatcertain factors may influence the dosage and timing required toeffectively treat a subject, including but not limited to the severityof the disease or disorder, previous treatments, the general healthand/or age of the subject, and other diseases present. Moreover,treatment of a subject with a therapeutically effective amount of afusion protein can include a single treatment or can include a series oftreatments.

Generally, partially and fully human fusion proteins are expected tohave a longer half-life within the human body are used for treatment ofhumans. Accordingly, lower dosages and less frequent administration isoften possible. Modifications such as lipidation can be used tostabilize a fusion protein and to enhance uptake and tissue penetration(e.g., into the brain). A method for lipidation of antibodies isdescribed by Cruikshank et al. (1997, J. Acquired Immune DeficiencySyndromes and Human Retrovirology 14:193) and can be adapted for usewith fusion proteins. Another method for increasing stability is toconjugate the fusion protein with human serum albumin

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Treatment Methods and Compositions

Provided herein are methods and compositions for both prophylactic andtherapeutic methods of treating a subject at risk of (or susceptible to)a disorder or having a disorder associated with a pathogen, e.g., asdescribed herein, including gonorrhea, meningococcal meningitis orsepsis, influenza, pertussis (whooping cough), pneumonia, pseudomonasinfection, or a yeast infection, as a result of infection, e.g. with N.gonorrhoeae, N. meningitidis, P. aeruginosa, H. influenzae, B.pertussis, S. pneumonia or C. albicans.

As used herein, the term “treatment” is defined as the application oradministration of a therapeutic agent (e.g., an agent comprising afusion protein) to a patient, or application or administration of atherapeutic agent to an isolated tissue or cell line from a patient, whohas a disease, a symptom of disease or a predisposition toward adisease, with the purpose to cure, heal, alleviate, relieve, alter,remedy, ameliorate, improve or affect the disease, the symptoms ofdisease or the predisposition toward disease. A therapeutic agent can bea fusion protein, a recombinant nucleic acid encoding a fusion protein,or a fusion protein that has been modified as described herein.

Pathogens that can be targeted using the fusion proteins describedherein include microbes, e.g., gram-positive and gram-negative bacteria,including, but not limited to, Pseudomonas aeruginosa, Streptococcuspneumoniae, Escherichia coli, Salmonella typhimurium, Neisseriameningitidis, Neisseria gonorrhoeae, Haemophilus influenzae andStaphylococcus aureus; fungi such as Aspergillus fumigatus, Candidaalbicans, spirochetes including Borrelia burgdorferi, and parasitesincluding Plasmodium falciparum. Any pathogen that binds the complementinhibitor factor H (FH) to evade complement-dependent killing can betargeted with a fusion protein as described herein.

A fusion protein can be delivered to a subject at risk for developing adisorder (e.g., after exposure to a biological weapon (e.g., Francisellatularensis (causes tularemia)) or a microbe that can cause illness, orbefore major surgery) or to treat an existing condition. Fusion proteinscan be delivered using methods known in the art, for example,systemically, or by direct delivery to a desired site such as joint orother area of a subject's body in which it is desirable to inhibit apathogen-related response such as inflammation, e.g., by injection orinhalation, e.g., of an aerosol; delivery by an aerosol may beparticularly useful in the case of exposure to an airborne pathogen.

Fusion proteins can also be delivered using a recombinant particle suchas a recombinant adenovirus containing an expressible nucleic acidsequence encoding the fusion protein. Such methods are known in the art(e.g., U.S. Pat. No. 5,998,598).

The fusion proteins described herein can also be used for thepreparation of a medicament for use in any of the methods of treatmentdescribed herein.

Liquid Purification Therapy

The methods of treating disorders associated with a pathogen asdescribed herein include the use of liquid, e.g., blood, purificationmethods. These methods can include temporarily removing blood from asubject, treating the blood with a fusion protein to remove soluble FHligands and pathogens, and returning the blood to the subject. Generalmethods for performing such purifications (sometimes referred to as“apheresis”) are known in the art, and typically involve passing theblood over a column or other device to extract a selected impurity, see,e.g., U.S. Pat. No. 6,569,112 (Strahilevitz); Asahi et al., TherapeuticApheresis 7(1):74-77(5), 2003; Hout et al., ASAIO J., 46(6):702-206,2000; Matsuo et al., Therapeutic Apheresis and Dialysis 8(3):194, 2004.These methods can be adapted for use in the present method. For example,a column or solid substrate including the fusion protein can beconstructed using methods known in the art, and the blood can be passedthrough it, removing a substantial amount of the FH ligands and/orpathogens present in the blood.

Alternatively, a collectible substrate, e.g., beads, e.g., magneticbeads, can be coated with the fusion protein, and the blood can be mixedwith the beads, and the beads then extracted to removed the FH ligandsand pathogens. In some embodiments, the blood is separated into itscomponents before being passed over the column or contacted with thebeads. In some embodiments, the methods can be used to remove FH ligandsand pathogens from the blood, by using a column or other collectiblesubstrate with covalently linked fusion proteins, which will pull FHligands and pathogens out of the blood. In some embodiments, more thanone type of fusion protein is used, and more than one type of FH ligandor pathogen is removed.

One of skill in the art will appreciate that these methods and otherknown fluid, e.g., liquid or gas, collection and filtering methods canalso be adapted to include the fusion proteins described herein for usein purifying liquids other than blood, e.g., water or any beverage, ormedia for use in culturing cells, as well as gases, such as air.

Methods of Diagnosis

Fusion proteins as described herein can be used for diagnostic purposes.Thus, included herein are methods for diagnosing a disorder associatedwith a FH-binding pathogen. The methods include obtaining a sample froma subject, contacting the sample with a fusion protein as describedherein under conditions sufficient to allow the fusion protein andpathogen to form complexes, and evaluating the presence and/or level ofa pathogen that binds to FH in the sample by detecting the complexes.The methods can also include comparing the presence and/or level withone or more references, e.g., a control reference that represents anormal level of FH-binding pathogen, e.g., a level in an unaffectedsubject (typically non-detectable), and/or a disease reference thatrepresents a level of FH ligand or pathogen, associated with thedisorder, e.g., a level in a subject having a disorder associated withthe pathogen. The presence and/or level of a protein can be evaluatedusing methods described herein, or other methods known in the art.

In some embodiments, the presence and/or level of FH-binding pathogen iscomparable to the presence and/or level of FH-binding pathogen in thedisease reference, and if the subject also has one or more symptomsassociated with a pathogen associated disorder, then the subject has apathogen associated disorder. In some embodiments, the subject has noovert signs or symptoms of a pathogen associated disorder, but thepresence and/or level of FH-binding pathogen is comparable to thepresence and/or level of FH-binding pathogen in the disease reference,then the subject has a pathogen associated disorder. In someembodiments, the sample includes a biological fluid, e.g., blood, semen,urine, and/or cerebrospinal fluid. In some embodiments, once it has beendetermined that a person has a pathogen associated disorder, then atreatment, e.g., as known in the art or as described herein, can beadministered.

Also included herein are methods for detecting a FH-binding pathogen inbiological or other samples, e.g., fluids such as blood, cell culturemedia, beverages, water, or air. The methods include obtaining a sample,and evaluating the presence and/or level of the FH-binding pathogen inthe sample using an assay described herein, e.g., an assay that detectsthe presence and/or level of FH-binding pathogen in the sample bydetecting the presence of a fusion protein/FH-binding pathogen complex.In some embodiments, the methods include comparing the presence and/orlevel with one or more references, e.g., a control reference thatrepresents a preselected level, e.g., a level above which the fluid isunsafe to use. These methods can be used in place of, or in addition to,e.g., Limulus amoebocyte lysate assays, which have limited use in blood(see, e.g., Hurley, Clinical Microbiology Reviews, 8(2):268-292 (1995).

In some embodiments, the sample is from a subject, and the presence ofFH-binding pathogen in the sample indicates that the subject has apathogen-associated disorder. These methods have the advantage thatFH-binding pathogens from a wide variety of pathogens may be detected,as opposed to methods such as PCR-based methods that may only detect oneor a subset of pathogens. In some embodiments, the assay is a simpleyes/no assay, and the results indicate that FH-binding pathogen ispresent in an unacceptable level. In some embodiments, the assayindicates what level of FH-binding pathogen is present.

Kits

Kits based on the fusion protein compounds described herein can bedeveloped and used, e.g., to screen biological fluids from infected(septic) patients, body fluids, or water or food, to name a fewapplications. The fusion proteins described herein can be used to detecta broad range of microorganisms including mycobacteria and fungi and socan be provided as reagents in a kit for detecting the presence of suchmicroorganisms.

A kit containing a fusion protein can include one or more types offusion proteins and a standard. The fusion protein can be packaged in asuitable container. The kit can also include instructions for using thekit to detect the presence of a pathogen, e.g., a microorganism or aclass or microorganisms.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Materials and Methods

The following materials and methods were used in the Examples set forthe below.

Bacterial Strains.

N. gonorrhoeae strains used in this study and their relevantcharacteristics are listed in Table 1. Strains from the sexuallytransmitted infection clinic in Nanjing, China were a randomly selectedconvenience sample. N. gonorrhoeae multiantigen sequence type (NG-MAST)was determined by DNA sequencing of variable regions of porB and tbpB,as previously described (30). An opacity protein (Opa)-negative mutantderivative of strain FA1090 where all 11 opa genes were inactivated wasprovided by Dr. Janne G. Cannon (University of North Carolina, ChapelHill, N.C., USA) (31). Bacteria that had been grown on chocolate agarsupplemented with IsoVitalex® in an atmosphere enriched with 5% CO₂ for˜15 h at 37° C. were suspended in gonococcal liquid media and grown tothe mid-log phase, as described previously (32). Sialylation ofgonococcal LOS was achieved by adding CMP-Neu5Ac to a finalconcentration of 2 ug/ml to the growth media. Bacteria were washed andsuspended in Hanks' balanced salt solution (HBSS) containing 0.15 mMCaCl₂ and 1 mM MgCl₂ (HBSS′) for use in binding and bactericidal assays.

TABLE 1 N. gonorrhoeae strains used in this study NG-MAST sequenceCeftriaxone Strain type MIC (ug/ml) Reference F62 915  NT^(A) (A) 252ND^(C) NT (B) MS11 4813 0.032 (C) FA1090 773 0.004 (D) FA1090 Opa⁻ 773NT (E) CTXr (Spain) 1407   2 (resistant) (F) H041 4220 2-4 (resistant)(G) NJ-1^(B) 3366 0.023 (H) NJ-11 270 0.047 (H) NJ-15 New #1^(D) 0.047(H) NJ-19 New #2^(D) 0.032 (H) NJ-26 New #3^(D) 0.032 (H) NJ-27 23180.064 (H) NJ-36 1766 0.032 (H) NJ-44 8736 0.125 (elevated (H) MIC toCRO) NJ-48 2008 0.125 (elevated (H) MIC to CRO) NJ-60 3289 0.380(resistant) (H) ^(A)NT, not tested; although an MIC was not obtained,all strains were susceptible to ceftriaxone by the disk-diffusion method^(B)NJ- strains were a random convenience sample from a collection of 64isolates obtained from men with gonorrhea from an STD clinic in Nanjing,China (8). Ceftriaxone susceptibility was performed by the tested byagar dilution method (8) ^(C)ND, not determined. ^(D)All new NG-MASTsequence types are distinct from each other having distinct PorB and/orTbpB loci. These sequences have been submitted for NG-MAST alleleassignment.

REFERENCES FOR TABLE 1

-   A. Shafer, W. M., K. Joiner, L. F. Guymon, M. S. Cohen, and P. F.    Sparling. 1984. Serum sensitivity of Neisseria gonorrhoeae: the role    of lipopolysaccharide. J Infect Dis 149:175-183.-   B. McQuillen, D. P., S. Gulati, S. Ram, A. K. Turner, D. B.    Jani, T. C. Heeren, and P. A. Rice. 1999. Complement processing and    immunoglobulin binding to Neisseria gonorrhoeae determined in vitro    simulates in vivo effects. J Infect Dis 179:124-135.-   C. Schneider, H., J. M. Griffiss, J. W. Boslego, P. J.    Hitchcock, K. M. Zahos, and M. A. Apicella. 1991. Expression of    paragloboside-like lipooligosaccharides may be a necessary component    of gonococcal pathogenesis in men. J Exp Med 174:1601-1605.-   D. Hitchcock, P. J., S. F. Hayes, L. W. Mayer, W. M. Shafer,    and S. L. Tessier. 1985. Analyses of gonococcal H.8 antigen: surface    location, inter- and intrastrain electrophoretic heterogeneity, and    unusual two-dimensional electrophoretic characteristics. J. Exp.    Med. 162:2017-2034.-   E. Lewis, L. A., S. Ram, A. Prasad, S. Gulati, S. Getzlaff, A. M.    Blom, U. Vogel, and P. A. Rice. 2008. Defining targets for    complement components C4b and C3b on the pathogenic neisseriae.    Infect Immun 76:339-350.-   F. Camara, J., J. Serra, J. Ayats, T. Bastida, D. Carnicer-Pont, A.    Andreu, and C. Ardanuy. 2012. Molecular characterization of two    high-level ceftriaxone-resistant Neisseria gonorrhoeae isolates    detected in Catalonia, Spain. J Antimicrob Chemother 67:1858-1860.-   G. Ohnishi, M., D. Golparian, K. Shimuta, T. Saika, S. Hoshina, K.    Iwasaku, S. Nakayama, J. Kitawaki, and M. Unemo. 2011. Is Neisseria    gonorrhoeae initiating a future era of untreatable gonorrhea?:    detailed characterization of the first strain with high-level    resistance to ceftriaxone. Antimicrob Agents Chemother 55:3538-3545.-   H. Li, S., X. Su, W. Le, F. Jiang, B. Wang, and P. A. Rice. 2014.    Antimicrobial susceptibility of Neisseria gonorrhoeae isolates from    symptomatic men attending the Nanjing sexually transmitted diseases    clinic (2011 inverted question mark2012): genetic characteristics of    isolates with reduced sensitivity to ceftriaxone. BMC Infect Dis    14:622.

Normal Human Serum (NHS).

Serum was obtained from normal healthy adult volunteers with no historyof gonococcal or meningococcal infection who provided informed consent.Participation was approved by the University of MassachusettsInstitutional Review Board for the protection of human subjects. Serumwas obtained by allowing blood to clot at 25° C. for 30 min followed bycentrifugation at 1500 g for 20 min at 4° C. Sera were pooled and storedat −70° C.

IgG and IgM Depleted Human Serum (Human Complement).

To study the effects of the FH18-20/Fc proteins without confounding bynatural anti-gonococcal antibodies present in NHS, we depleted IgG andIgM from freshly collected human serum, as described previously (33).Briefly, EDTA (final concentration 10 mM) and NaCl (final concentration1 M) were added to freshly prepared human serum and treated sera waspassed first over anti-human IgM agarose (Sigma), followed by passagethrough protein G-Sepharose; both columns were equilibrated in PBScontaining 10 mM EDTA and 1 M NaCl. NaCl was added to minimize C1qdepletion during passage of serum through the anti-human IgM column. Theflow-through was collected, spin concentrated and dialyzed againstPBS/0.1 mM EDTA to its original volume using a 10-kDa cutoff AmiconUltra-15 centrifugal filter device (Millipore, Bedford, Mass.),sterilized by passage through a 0.22-μm filter (Millipore), aliquotedand stored at −70° C. Hemolytic activity was confirmed using a totalcomplement hemolytic plate assay (The Binding Site Inc., Birmingham,U.K). Depletion of IgG and IgM was confirmed by dot-blot assays. In someexperiments, complement activity of serum was destroyed by heating serumat 56° C. for 1 h.

Expression and Purification of FH/Fc Fusion Proteins.

Cloning, expression and purification of a chimeric protein comprisinghuman FH (HuFH) domains 18-20 fused to mouse IgG2a Fc has been describedpreviously (23). Briefly, the DNA encoding FH domains 18-20 was clonedinto AscI-NotI sites of eukaryotic expression vector pCDNA3 containingthe sequence encoding mouse IgG2a Fc (34). We created four HuFH18-20/Fcmutants using the Quickchange site-directed mutagenesis kit (AgilentTechnologies) according to the manufacturer's instructions with primersD1119G, R1182S, W1183R and R1215G (Table 2). Mouse IgG2a Fc was replacedby human IgG1 Fc as follows. FH domains 18-20 were amplified usingprimers FH18EcoRI and FH20hIgGloverlapR, and human IgG1Fc (Invivogen)was amplified with primers FH20hIgGloverlapF and HIgG1NheI (Table 2).The PCR products were then fused together by overlap extension PCR usingprimers FH18EcoRI and HIgG1NheI. The final PCR product encoding FH18-20fused to hIgG1 was digested with EcoRI and NheI and cloned intopFUSE-hIgG1-Fc2 (Invivogen). The resulting plasmids were verified by DNAsequencing and used to transiently transfect CHO cells using lipofectin(Life Technologies), according to the manufacturer's instructions. Mediafrom transfected cells was collected after 2 days and FH/Fc was purifiedby passage over protein A agarose. Mass was determined by Coomassie Bluestaining of proteins separated by SDS-PAGE and protein concentrationswere determined using the BCA protein Assay kit (Pierce).

TABLE 2 Primers used for construction of FH/Fc fusion proteins PrimersSequence SEQ ID NO: D1119G 5′-CACCTATTGACAATGGGGGCATTACTTCATTCCCGTT-3′SEQ ID NO: 7 R1182S 5′- SEQ ID NO: 8ATTATGGAAAATTATAACATAGCATTAAGCTGGACAGCC AAACAGAAG-3′ W1183R 5′-SEQ ID NO: 9 AAAATTATAACATAGCATTAAGGAGGACAGCCAAACAGA AGCTTTAT-3′ R1215G5′-CACGTTCTCACACATTGGGAACAACATGTTGGGAT-3′ SEQ ID NO: 10 FH18EcoRI5′-GAATTCGTGTGTGAATCCGCCCACAGTAC-3′ SEQ ID NO: 11 FH20hIgG15′-AGCCCAAATCTTG TGACAAAACTCACACATGCCCA-3′ SEQ ID NO: 12 overlapFFH20hIgG1 5′-GCCGCGGGGGGCGAGCCCAAATCTTGTGACAA-3′ SEQ ID NO: 13 overlapRHIgG1NheI 5′-CGGGTAAATGAGTGCTAGCTGG-3′ SEQ ID NO: 14

Antibodies.

Sheep anti-human C3c-FITC was from AbD Serotec (cat. #AHP031F),anti-mouse IgG FITC and anti-human IgG FITC were from Sigma. Bothantibodies (Abs) were used at a dilution of 1:200 in HBSS' and 1% bovineserum albumin (BSA) (HBSS⁺⁺/BSA) in flow cytometry assays.

Flow Cytometry.

Binding of FH/Fc to bacteria and C3 fragments deposited on bacteria weremeasured by flow cytometry as described previously (35). Data wereacquired on a LSRII flow cytometer and data were analyzed using FlowJosoftware.

Serum Bactericidal Assay.

Serum bactericidal assays were performed as described previously (36).Bacteria that had been harvested from an overnight culture on chocolateagar plates were grown in gonococcal liquid media supplemented withCMP-Neu5Ac (2 ug/ml) from an OD_(600 nm) of ˜0.1 to the mid-log phase(OD_(600 nm) ˜0.25). Approximately 2000 colony forming units (CFUs) ofN. gonorrhoeae were incubated with human complement in the presence orthe absence of the FH/Fc fusion protein (concentration indicated witheach experiment). The final volume of the bactericidal reaction mixturewas 150 ul. Aliquots of 25-ul reaction mixtures were plated ontochocolate agar in duplicate at the beginning of the assay (to) and againafter incubation at 37° C. for 30 min (t₃₀). Survival was calculated asthe number of viable colonies at t₃₀ relative to to.

Hemolytic Assay.

Lysis of human erythrocytes was measured using a method similar to onedescribed previously (37). Freshly isolated human red blood cells (RBCs)(5×10⁶) were incubated with 7 ug/ml anti-CD59 monoclonal antibody (mAb)(clone MEM43; Abcam) at 4° C. for 20 min and then mixed, on ice, withNHS derived from the homologous donor (final NHS concentration 40%),gelatin veronal buffer (GVB), 5 mM MgCl₂ and 5 mM EGTA and the indicatedconcentrations of FH/Fc. The mixture was then transferred to a 37° C.water bath and incubated for 20 min. GVB-EDTA (200 ul) was added to afinal concentration of 10 mM EDTA to block further complement activationand the samples were immediately centrifuged at 4° C. and theOD_(410 nm) of the supernatants was determined. Background lysis(anti-CD59-treated RBCs plus buffer alone) was subtracted from eachreading and the results were expressed as OD_(410 nm).

Opsonophagocytosis Assay.

Heparinized venous blood was obtained from a healthy adult volunteer inaccordance with a protocol approved by the Institutional Review Boardfor the protection of human subjects at the University of Massachusetts.Polymorphonuclear leukocytes (PMNs) were isolated using Mono-Poly®resolving medium (MP Biomedicals, LLC) according to the manufacturer'sinstructions. Isolated PMNs were washed and suspended in HBSS withoutadded divalent cations, counted, and diluted to 1×10⁷/ml inHEPES-buffered RPMI-1640 medium supplemented with L-glutamine and 1%heat-inactivated fetal bovine serum (FBS). To measure survival ofgonococci in the presence of PMNs, an Opacity protein (Opa)-negativemutant of N. gonorrhoeae strain FA1090 that was grown in mediacontaining 2 ug/ml CMP-Neu5Ac to sialylated LOS was added to 1×10⁶ PMNsat an multiplicity of infection (MOI) of 10 (10 bacteria to 1 PMN).Opa-negative N. gonorrhoeae was used because select Opa proteins serveas ligands for human carcinoembryonic antigen-related cell adhesionmolecule 3 (CEACAM3) that is expressed by PMNs, which results inphagocytosis (38). FHD1119G/HuFc was added at a concentration of 16.7μg/ml, followed by 10% human complement (prepared as described above).Bacteria plus PMNs and 10% NHS (Ab intact) was used as a positivecontrol for killing. The reaction mixtures were incubated for 60 min at37° C. in a shaking water bath. Bacteria were serially diluted andplated at 0 and 60 min on chocolate agar plates. Percent survival ofgonococci in each reaction was calculated as a ratio of CFUs at 60 minto CFUs at the start of the assay (0 min).

Mouse Vaginal Colonization Model.

Use of animals in this study was performed in strict accordance with therecommendations in the Guide for the Care and Use of Laboratory Animalsof the National Institutes of Health. The protocol was approved by theInstitutional Animal Care and Use Committee (IACUC) at the University ofMassachusetts Medical School. Female BALB/c mice 5-6 weeks of age(Jackson Laboratories) in the diestrus phase of the estrous cycle werestarted on treatment (that day) with 0.5 mg Premarin (Pfizer) in 200 μlof water given subcutaneously on each of three days; −2, 0 and +2 days(before, the day of and after inoculation) to prolong the estrus phaseof the cycle and promote susceptibility to Ng infection. Antibiotics(vancomycin, colistin, neomycin, trimethoprim and streptomycin)ineffective against Ng were also used to reduce competitive microflora(39). Mice (n=26) were then infected with 1.5×10⁶ CFU of strain F62. Onegroup of mice (n=14) was treated with 12 FHD1119G/mouse IgG2a Fc (1.5mg/ml in PBS) daily intravaginally while the remaining 12 mice weregiven a corresponding volume of PBS (vehicle controls). We used aconstruct containing mouse IgG2a Fc (and opposed to human IgG1 Fc usedin the bactericidal and opsonophagocytosis assays) to maintain speciescongruity between Fc and its cognate FcR. Initial experiments withsystemic administration of FH/Fc to wild-type mice resulted in thegeneration of anti-FH Ab, which led us to administer the therapeuticlocally. Finally, we administered 10 μg of CMP-Neu5Ac locally to eachmouse daily along with the FH/Fc or PBS control to ensure LOSsubstitution with Neu5Ac. This is because mice, but not humans, possessan enzyme called CMP-N-acetylneuraminic acid hydroxylase (CMAH) thatconverts CMP-Neu5Ac to CMP-N-glycolylneuraminic acid (CMP-Neu5Gc)(40-42). CMAH activity, and therefore the relative amounts of these twoCMP-sialic acids, varies across tissues (43). Thus, while mice expressboth CMP-Neu5Ac and CMP-Neu5Gc, humans make only CMP-Neu5Ac. Both theseCMP-sialic acids can serve as substrates for gonococcal LOSsialyltransferase, therefore gonococcal LNT LOS can be substituted witheither Neu5Ac or Neu5Gc ($$ REF). In contrast, a mutation in humansresults in inactivation of CMAH (41) and as a result gonococcal LOS inhumans is exclusively substituted with Neu5Ac. Administering CMP-Neu5Acwould result in more ‘human-like’ LNT LOS substitution with Neu5Ac.

Statistics.

Experiments that compared clearance of N. gonorrhoeae in independentgroups of mice estimated and tested three characteristics of the data(44): Time to clearance, longitudinal trends in mean log₁₀ CFU and thecumulative CFU as area under the curve (AUC). Statistical analyses wereperformed using mice that initially yielded bacterial colonies on Days 1and/or 2. Median time to clearance was estimated using Kaplan-Meiersurvival curves; the times to clearance were compared between groupsusing a log-rank test. Mean log₁₀ CFU trends over time were comparedbetween groups using a linear mixed model with mouse as the randomeffect using both a random intercept and a random slope. A cubicfunction in time was determined to provide the best fit; random slopeswere linear in time. A likelihood ratio test was used to compare nestedmodels (with and without the interaction term of group and time) to testwhether the trend differed over time between the two groups. The meanAUC (log₁₀ CFU versus time) was computed for each mouse to estimate thebacterial burden over time (cumulative infection); the means under thecurves were compared between groups using the nonparametric rank sumtest because distributions were skewed or kurtotic.

Example 1. Selection of the FHD1119G/Fc as the Lead FH/Fc TherapeuticMolecule

We showed previously that a chimeric molecule comprising FH domains 18,19 and 20 fused to murine Fc bound to and mediated complement-dependentkilling of N. gonorrhoeae strains F62 and 252 (27). However, theC-terminal domains of FH (domains 19 and 20) can bind to C3b/C3d (19,45), heparin/heparan sulfate-containing surfaces (46), and endothelialcells (47) and protects host cells from complement attack. Thus, if leftunmodified in the context of FH/Fc, the C-terminal domains of FH(domains 19 and 20) will compete with binding and function of thefull-length FH on human cells. Competition for the binding and functionof full-length FH by a recombinant FH molecules comprising domains 19and 20 was shown on RBCs treated with anti-CD59 (erythrocytes treatedwith anti-CD59 rely on binding of FH to regulate complement activationand hemolysis) (37). Therefore, we sought to define mutations in FH18-20that eliminated complement activation on host cells while maintainingthe ability to bind to and mediate killing of N. gonorrhoeae.

Atypical hemolytic uremic syndrome (aHUS) is a condition that resultsfrom ‘over-activity’ of the alternative pathway of complement. Mutationsof FH that affects FH binding to glycosaminoglycans and/or C3 fragmentson host cells are important causes for the development of aHUS (reviewedin references (48-50)). Our choice of mutations was guided by some ofthe aHUS mutations in FH domains 19 and 20 that were characterized byFerreira et al (37). They introduced these mutations into recombinantmolecules that comprised FH domains 19 and 20, and examined whether themutant molecules could block full-length human FH from protectinganti-CD59-treated human RBCs from complement-mediated hemolysis (37). Asexpected, the wild-type FH 19-20 out-competed the full-length FH andresulted in hemolysis. Four mutant molecules, D1119G (domain 19),R1182S, W1183R and R1215G (the latter three in domain 20) did notinterfere with the normal function of native FH (37), which led us tofocus on these four aHUS mutations. FH18-20/murine IgG2a Fc proteinsthat contained these individual mutations were expressed in CHO cellsand purified from tissue culture supernatants; the molecular masses andpurity of the proteins was determined by SDS-PAGE stained with CoomassieBlue (data not shown). Bacteria were grown in media containingCMP-Neu5Ac, which results in sialylation of LNT LOS, similar tosialylation that occurs in vivo (20). We compared binding of FH18-20/Fcmutant proteins to sialylated gonococci with binding of the wild-type(WT) FH18-20/Fc (FIG. 1A). Two mutant proteins, FHD1119G/Fc andFHR1182S/Fc showed similar binding to sialylated N. gonorrhoeae strainF62 compared to the wild-type protein (FH18-20/Fc). The FH/Fc moleculesbearing the W1183R or R1215G mutations, showed weak and no binding tosialylated strain F62 respectively, (FIG. 1A). FH/Fc mutated proteinswere tested for complement-dependent killing of sialylated strain F62(FIG. 1B, left graph). Consistent with our previous work (27), thewild-type molecule (FH18-20/Fc) killed strain F62 in a dose-responsivemanner (FIG. 1B); FHD1119G/Fc and FHR1182S/Fc also both killedsialylated strain F62. Neither FHR1183R/Fc or FHR1215/Fc, killedsialylated strain F62 when used at maximal concentration (6.7 ug/ml).

The killing curves, in particular for the wild-type molecule and theD1119G mutant for sialylated strain F62, were steep—i.e., smalldifferences in FH/Fc concentration resulted in dramatic increases incomplement-dependent killing. To ascertain superiority of function ofFHD1119G/Fc mutant over FHR1182S/Fc mutant, we used strain 252 that isintrinsically more resistant to killing by complement than F62 (36, 51).As shown in (FIG. 1B, right graph), only the wild-type molecule and theD1119G mutant possessed activity against sialylated strain 252. Thus,FHD1119G/Fc represented our lead molecule and was studied further.

Example 2. FHD1119G/Fc does not Cause Hemolysis

Having identified the D1119G mutant (FHD1119G/Fc) as the molecule withthe best bactericidal activity among the mutants tested, we next askedwhether this mutation eliminated toxicity to host cells, as measured bythe human RBC lysis assay described by Ferreira et al (37).Complement-mediated lysis of human RBCs was measured in the presence ofNHS and FHD1119G/Fc or the control wild type FH18-20/Fc (FIG. 2). TwoFHD1119G/Fc chimeric molecules were tested—one that contained mouseIgG2a Fc and a second containing human IgG1 Fc. The latter was developedfor use in studies with human PMNs (see below) and in anticipation ofpossible use as a therapeutic antimicrobial in humans. FIG. 2 showslysis of RBCs when mouse FH18-20/Fc (WT) protein was added to thereaction mixture (positive control); FHD1119G/Fc proteins (mouse orhuman Fc) did not cause measurable lysis over baseline levels (controlswith buffer alone) at any concentration tested (0-66.7 ug/ml).FHD1119G/mouse IgG2a Fc and FHD1119G/human IgG1 Fc exhibited similarbactericidal activities against three different strains of N.gonorrhoeae (FIG. 8). FHD1119G/human IgG1 Fc was used in subsequentexperiments.

Example 3. Binding of FHD1119G/Fc to N. gonorrhoeae

A clinically useful anti-bacterial immunotherapeutic should possessactivity against a wide repertoire of clinically relevant strains. Wenext tested binding of FHD1119G/human IgG1 Fc to 15 clinical isolates ofN. gonorrhoeae (listed in Table 1), including three contemporaryceftriaxone-resistant isolates: CTXr(Sp) (4), H041 (7), and NJ-60 (52)and two isolates with elevated MICs to ceftriaxone (NJ-44 and NJ-48)(52). All strains were grown in media supplemented with 2 ug/mlCMP-Neu5Ac to sialylate LOS, as occurs in vivo (20, 21). Although thebinding of FHD1119G/HuFc varied across strains, binding was seen to allsialylated strains that were tested in a flow cytometry assay (≈6 to346-fold fluorescence increase over control values; FIG. 3).Representative histogram tracings from a flow cytometry experiment areshown in FIG. 9.

Example 4. Bactericidal Activity of D1119G/Fc Against N. gonorrhoeae

FHD1119G/Fc was tested for complement dependent killing of the 15sialylated clinical isolates of N. gonorrhoeae (FIG. 4). Ten of 15strains showed from 0% to <50% survival (50%-100% killing) compared tobaseline survival (bacteria plus human complement (normal human serumdepleted of IgG and IgM) alone) (FIG. 4), while the remaining 5 strains(FA1090, CTXr (Sp), NJ-11, NJ-19 and NJ-26) survived >50% in thepresence of FHD1119G/Fc (marked with an “#” in FIG. 4). It is worthnoting that the amount of binding of FHD1119G/Fc did not correlate withbacterial killing.

Example 5. C3 Fragment Deposition Mediated by FHD1119G/Fc on N.gonorrhoeae Resistant to Direct Complement-Dependent Lysis

Complement-dependent opsonophagocytosis may also contribute to clearanceof gonococci in humans. We sought to determine whether the 5 isolatesthat resisted direct killing by complement (mediated by insertion of themembrane attack complex [C5b-9]), had also resisted deposition of C3.Sialylated bacteria were incubated either with human complement alone,or complement plus FHD1119G/Fc; C3 fragments deposited on bacteria weremeasured by flow cytometry (FIG. 5). Minimal C3 was deposited on thesestrains in the presence of complement alone (compared to baselineantibody conjugate control levels). Addition of FHD1119G/Fc markedlyincreased C3 fragment deposition. Increases ranged from 13- to 88-foldabove levels seen with complement alone. These data suggest thatresistance of these strains to direct complement-dependent killing maybe the result of a block in complement function distal to C3 deposition.

Example 6. FHD1119G/Fc Enhances Complement-Dependent Killing by PMNs

Having shown that FHD1119G/Fc augments C3 deposition on gonococci, weasked whether it could also facilitate opsonophagocytic killing by humanPMNs. Select gonococcal opacity proteins (Opa) can engage human CEACAMs(38, 53). CEACAM3 that is expressed by human PMNs can facilitate uptakeand killing of gonococci through Opa in a complement and FcR independentmanner (38). To eliminate Opa-CEACAM3 interactions, we used anOpa-negative derivative of strain FA1090 where all 11 opa genes had beeninactivated (31). Similar to wild-type FA1090, the isogenic Opa-negative(Opa) mutant of FA1090 also resisted direct complement-dependent killing(>100% survival; data not shown). FHD1119G/Fc increased killing ofFA1090 Opa-negative in the presence of active complement and PMNs (FIG.6). Complement alone or FHD1119G/Fc alone did not facilitatePMN-dependent killing of bacteria, indicating that both complement andFc were required for opsonophagocytic killing of Opa-negative gonococciby human PMNs in vitro.

Example 7. FHD1119G/Mouse IgG2a Fc Decreases the Burden and Duration ofGonococcal Infection in the Mouse Vaginal Colonization Model

The efficacy of FHD1119G/mouse IgG2a Fc was tested in the mouse vaginalcolonization model. The rationale for the use of mouse Fc, intravaginaladministration and the concomitant use of CMP-Neu5Ac have all beendiscussed above in the Materials and Methods. Mice were infected withstrain F62 and given either 12 μg of D1119G/Fc daily intravaginally for7 days (n=14 mice) or PBS as a vehicle control (n=12). As shown in FIG.7, the group that received FHD1119G/mouse IgG2a Fc cleared the infectionfaster (FIG. 7A; median time to clearance was 5 days, versus 7 days inthe control group; P=0.05; clearance times were compared between groupsusing a log-rank test). Mixed model analysis indicated significantdifferences in colonization trends between the two groups comparingD1119G/Fc and PBS-treated groups (P<0.0001; FIG. 7B). A significantdifference in the Mean Areas Under the Curve (mean AUCs) (log 10 CFUversus time) between the treated and control groups was also observed(P=0.012; FIG. 7C).

Example 8. FHD1119G/Human IgG1 Fc Decreases the Burden and Duration ofGonococcal Infection of Strains FA1090 and H041 in the Mouse VaginalColonization Model

To determine whether FHD1119G/human IgG1 Fc and FHD1119G/mouse IgG2a Fchad comparable bactericidal activities, the survival of sialylated N.gonorrhoeae F62, H041 and NJ-60 in increasing concentrations of FHD1119Gfused to either human IgG1 Fc or mouse IgG2a Fc was determined followingthe addition of 17% (v/v) pooled normal human serum (NHS; not depletedof natural Ab). FHD1119G/human IgG1 Fc and FHD1119G/mouse IgG2a Fc havecomparable bactericidal activities at all concentrations tested (FIG.8). Next, the binding of FHD1119G/human IgG1 Fc to 15 clinical isolatesof N. gonorrhoeae sialylated was evaluated in vitro. Each of the 15isolates bound FH/Fc (FIG. 9).

The efficacy of FHD1119G/human IgG1 Fc was tested in the mouse vaginalcolonization model. Mice were infected with ˜10⁶ CFU of strain FA1090 orceftriaxone-resistant (CRO-R) strain H04 and were given either 10 μg ofFHD1119G/human IgG1 Fc daily intravaginally (n=10 mice) or PBS as avehicle control (n=10). As shown in FIG. 10, the groups that receivedFHD1119G/human IgG1 Fc cleared either infection faster (FIG. 10A; mediantime to clearance was 5 days, versus 9 days in the control group;P<0.0001; clearance times were compared between groups using Mantel-Coxanalysis; FIG. 11A; median time to clearance was 6 days, while none ofthe mice in the control group were cleared by 8 days; P<0.0001;clearance times were compared between groups using Mantel-Coxanalysis;). Two-way ANOVA indicated significant differences between thetwo groups comparing FHD1119G/human IgG1 Fc and PBS-treated groups (FIG.10B and FIG. 11B); the main effect for time, log 10 CFU and theinteraction between time and log 10 CFU were significant in bothinstances (P<0.0001); *, P<0.05; **, P<0.01; #, P<0.0001 by Sidak's testat the indicated time points). A significant difference in the MeanAreas Under the Curve (mean AUCs) (log 10 CFU versus time) between thetreated and control groups was also observed (P<0.0001; FIG. 10C andFIG. 11C).

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OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A fusion polypeptide consisting of Factor H (FH)domains 18-20 (FH18-20) linked via a linker to a human IgG1 Fc region,wherein the FH18-20 has a mutation at position 1119 in domain 19, andwherein the linker comprises at least two additional amino acids betweenthe FH domain and the human IgG1 Fc region.
 2. The fusion polypeptide ofclaim 1, wherein the linker comprises at least one glycine or onealanine.
 3. The fusion polypeptide of claim 1, wherein the linkercomprises GAAGG (SEQ ID NO: 1) or AAAGG (SEQ ID NO:2).
 4. The fusionpolypeptide of claim 1, wherein the FH18-20 has a mutation of D to G atposition 1119 in domain
 19. 5. A pharmaceutical composition comprisingthe fusion polypeptide of claim 1, and a pharmaceutically acceptablecarrier.
 6. A method of treating a disorder associated with a FactorH-binding pathogen in a subject, the method comprising administering atherapeutically effective amount of a fusion polypeptide of claim
 1. 7.The method of claim 6, wherein the disorder is a pathogen-associatedinfection or an inflammatory condition.
 8. The method of claim 6,wherein the pathogen is selected from the group consisting of bacteria,fungi, viruses, spirochetes, and parasites.
 9. The method of claim 8,wherein the bacterium is selected from the group consisting of P.aeruginosa, S. pneumoniae, Y. pestis, E. coli, S. typhimurium, N.meningitidis, N. gonorrhoeae, H. influenza and S. aureus; the fungus isselected from the group consisting of Aspergillus fumigatus, Candidaalbicans, and other zymosan-containing fungi; the spirochete is Borreliaburgdorferi or Treponema pallidum; or the parasite is Plasmodium bergheior Plasmodium falciparum.